Nitrilase from arabis alpina, its encoding gene, vector, recombinant bacterial strain and uses thereof

ABSTRACT

The disclosure provides a nitrilase from  Arabis alpina , which belongs to genus  Arabis , family brassicaceae. The disclosure further provides the encoding gene, vector, recombinant bacterial strain, and the application in the manufacturing of (S)-3-cyano-5-methylhexanoic acid. The wet resting cells containing nitrilase Aa-Nit can kinetically resolve racemic IBSN at 1.2 M with a 42% conversion rate in 15 hr and &gt;99% ee value. The disclosure provides a regio- and stereoselective method for the preparation of (S)-3-cyano-5-methylhexanoic acid. This method provides an atom economical, mild, environmental friendly industrial method to manufacture (S)-3-cyano-5-methylhexanoic acid.

This application claims the benefit of Chinese patent application No.201510535881.1, filed Aug. 27, 2015, the entirety of which isincorporated herein by reference.

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named “CCHMP0002US_ST25.txt”, created onAug. 22, 2016 and having a size of ˜5 kilobytes. The content of theaforementioned file is hereby incorporated by reference in its entirety.

1. Technical Area

This invention relates to a method of manufacturing(S)-3-cyano-5-methylhexanoic acid. It further relates to a nitrilasefrom Arabis alpina and the application in the manufacturing of(S)-3-cyano-5-methylhexanoic acid.

2. Background

Pregabalin (I, PGB), chemical name (S)-3-(aminomethyl)-5-methylhexanoicacid (I), is a 3-isobutyl substituted γ-aminobutanoic acid (Angew. Chem.Int. Ed. 2008, 47: 3500-3504). PGB is very effective in treatingepilepsy, diabetic neuropathy pain, and post-herpetic neuralgia pain. Itrequires lower dosages and less frequent administration, has fewer sideeffects, lasts longer, and is well tolerated. PGB has become ablockbuster in global pharmaceutical markets.

(S)-3-Cyano-5-methylhexanoic acid (II) is a key chiral intermediate thatcan be converted to PGB through hydrogenation. Pfizer, Inc. hasdeveloped a second-generation, enzymatic process for PGB, where2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester diethyl2-(1-cyano-3-methylbutyl)malonate was resolved through Lipolase®catalysis, decarboxylated, and hydrolyzed under basic condition toafford II (Org. Process Res. Dev. 2008, 12: 392-398). This processrequires decarboxylation and two hydrolysis steps, leading to low atomeconomy.

Nitrilase (EC 3.5.5.1) is an enzyme that can catalyze the hydrolysis ofnitriles to ammonia and the corresponding carboxylic acid. Nitrilase hasvery strict regio- and stereo-selectivity and shows high potential inthe manufacture of highly valuable active pharmaceutical ingredients.Pfizer's Xie reported that Arabidopsis thaliana nitrilase At-Nit1 couldregio- and stereo-selectively convert racemic 2-isobutylsuccinonitrile(ISBN) into II (J. Mol. Catal. B: Enzym. 2006, 41:75-80). This processenjoys a very high atom economy. However, nitrilase At-Nit1 has lowactivity and only provides II with 17.5% yield when the substrateconcentration was 150 mM (WO2005100580). Another enzyme NIT-102 couldonly provide II at 31.3% when the substrate was treated for 24 hr at 400mM (WO2005100580). This method is difficult to industrialize because ofthe long reaction time and low substrate concentration. Thus, it is verynecessary to find nitrilases with industrial potential for themanufacturing of II.

SUMMARY OF THE INVENTION

The goal of the current invention is to find a novel nitrilase that canregio- and stereoselectively convert ISBN into II. The nitrilase shouldshow higher catalytic activity and better tolerance to substrate so thatII could be manufactured in industrial scale. When at 400 mM, ISBNshould be converted to II at least 42%.

An embodiment of the current invention is a nitrilase (Aa-Nit) fromArabis alpina, which belongs to family brassicaceae. The nitrilase'samino acid sequence is shown in SEQ ID No. 1.

A further embodiment of the current invention covers any polypeptidesegments or mutants obtained through knockout, insertion, replacement ofone or more amino acid residues, as long as the sequence resembles 95%of SEQ ID No. 1.

Another embodiment of the current invention further covers a nitrilase'sencoding gene. To realize the heterologous expression of soluble Aa-Nitin E. coli, the nucleotide (SEQ ID No. 2) corresponding to the aminoacid sequence (SEQ ID No. 1) was synthesized through routine geneticengineering procedures.

Yet another embodiment of the current invention covers any nucleotidesegments obtained through knockout, insertion, replacement of one ormore nucleoside residues, as long as the sequence resembles 90% of SEQID No. 2.

Another embodiment of the current invention relates to a recombinantvector for the nitrilase encoding gene, and the recombinant geneticallyengineered bacterium obtained with the above-stated vector.

A further embodiment of the current invention relates to the method ofpreparing nitrilase Aa-Nit from the corresponding nitrilase gene. Thenitrilase gene containing vector is introduced into the host cells toobtain the genetically engineered bacteria. The bacteria are cultured toobtain nitrilase-containing cells. The procedure is as follows: (1)Nitrilase Aa-Nit's nucleotide sequence was obtained through gene miningtechnique. (2) The synthesized nitrilase gene segment is inserted intopEt28b vector to afford the recombinant plasmid. (3) The recombinantplasmid is introduced into the host cell, preferably E. coli BL21 (DE3)to afford the corresponding engineered strain. (4) The engineered strainis inoculated into LB medium, grown to log phase, and induced with0.1-0.2 mM IPTG at 28° C. for 12 hrs. (5) The cells are collected andthe target protein size and expression is verified by SDS-PAGEelectrophoresis (see FIG. 2).

Yet a further embodiment of the current invention relates to theapplication of nitrilase catalysis for the synthesis of II.Specifically, the engineered strain was fermented to obtainnitrilase-containing wet cells, which was used as the catalyst for thehydrolysis of racemic 2-isobutylsuccinimide in a pH 5.0-10.0 buffer(preferably in 100 mM, pH 8.0 Tris-HCl buffer) at 25-45° C. and stirredat 150 rpm (preferably at 30° C. and 150 rpm). After the hydrolysis iscomplete, II is isolated from the reaction mixture. The amount ofcatalyst used is 50 g/L of buffer based on the weight of the wet cells.The concentration of the substrate is 0.15-1.5 mol/L (preferably 0.7-1.2mol/ L).

The catalyst in the current invention is prepared as following: Thenitrilase-containing engineered strain, preferably E. coli BL21(DE3)/pET28b(+)-Aa-Nit, is inoculated into a liquid LB broth containing50 μg/mL of kanamycin and grown for 12 hr at 37° C. The cultured brothwas inoculated into a fresh liquid LB broth containing 50 μg/mL ofkanamycin at 2% (v/v). The strain was grown at 37° C. until the cellconcentration (OD₆₀₀) ca. 0.6 (0.4-0.8), and IPTG was then added to afinal concentration of 0.2 mM to induce the protein expression 28° C.for 12 hr. After centrifugation at 4° C., 12000 rpm for 5 min, the wetcells are collected as catalyst.

A further embodiment of the current invention relates to the separationand purification method of II. After the reaction, the reaction mixtureis centrifuged to remove E. coli. The supernatant is evaporated to ⅓ ofthe original volume. The temperature was maintained at 80° C. for 40 minto denature the proteins before the removal of the proteins throughcentrifugation. A preferred method of protein removal is through afurther vacuum filtration. The filtrate is extracted with ethyl acetate(2× volume). The aqueous layer is acidified with 2 M HCl to pH 4.0.Extraction with ethyl acetate (2 volumes), followed by evaporation ofethyl acetate on rotavap, affords II as an oil.

The current invention provides a region- and stereo-selective enzyme forthe manufacturing of II through hydrolysis of IBSN. The resting cellscontaining nitrilase Aa-Nit can kinetically resolve IBSN at 1.2 M withthe conversion rate of 42% in 15 hr and ee>99%. The current inventionprovides an atom economical, mild, environmental friendly industrialmethod to manufacture II.

ILLUSTRATIONS

FIG. 1. Nitrilase Aa-Nit catalyzed kinetic resolution of IBSN.

FIG. 2. SDS-PAGE analysis of Nitrilase Aa-Nit. M: Molecular weight ofproteins; 1: Induced expression of the target protein.

FIG. 3. Gas chromatography of nitrilase Aa-Nit catalyzed kineticresolution of IBSN.

FIG. 4. Optimization of the pH of the reaction system.

FIG. 5. Optimization of the temperature of the reaction system.

EXAMPLES

The following examples are for the illustration of the current inventionand in no way represents the scope of the current invention.

The main experimental materials were purchased from the followingsources:

E. coli host strain: E. coli BL21 (DE3) Invitrogen Expression vectorpEt-28b(+) Novagen Restriction endonucleases Xho I and Xba I FermentasT4 DNA ligase TaKaRa Kanamycin TaKaRa IPTG Promega DNA marker and stainGoldView TaKaRa DNA gel extraction kit Axygen PCR Clean-up kit AxygenPlasmid extraction kit Axygen

Example 1

Preparation of Nitrilase Aa-Nit

(1) Nitrilase Aa-Nit amino acid sequence and nucleic acid sequence. Anitrilase amino acid sequence (Genbank No. KFK44999.1) was obtained viascreening nitrilase gene sequence from protein database PDB and NCBI.The nitrilase comes from Arabis alpina, a plant belong to genus Arabis,family Brassicaceae. Based on the amino acid sequence of nitrilase,optimized codons from E. coli preferred codons, and the characteristicsof vector pET28b(+), restriction enzyme cutting sites Xho I and Xba Iwere selected. The nitrilase-coding nucleic acid (shown in SEQ ID No. 2)and the coded amino acid sequence (shown in SEQ ID No. 1) weresynthesized.

(2) Construction of recombinant strain. The nucleic acid segment wastreated with restriction endonucleases Xho I and Xba I and recovered.The recovered gene and commercial vector pET28b(+) (pre-treated withrestriction endonucleases Xho I and Xba I) were treated with T4 DNAligase for 16 hr at 16° C. to give Intracellular recombinant expressionvector pET28b(+)-Aa-Nit, which was introduced into E. coli BL21 (DE3)(Invitrogen), which was then spread onto a LB agar-plate containing 50μg/ml of kanamycin and grown overnight at 37° C. The strains grown onthe plate was randomly selected and the plasmid was extracted foragarose gel electrophoresis.

(3) Induced expression of nitrilase Aa-Nit. The recombinant geneticallyengineered E. coli BL21 (DE3)/pET28b(+)-Aa-Nit was inoculated into aliquid LB broth containing 50 μg/mL of kanamycin and grown for 12 hr at37° C. The LB broth was inoculated into a fresh liquid LB brothcontaining 50 μg/mL of kanamycin at 2% (v/v). The strain was grown at37° C. until the cell concentration (OD₆₀₀) reached 0.6 and IPTG wasthen added to a final concentration of 0.2 mM to induce the proteinexpression 28° C. for 12 hr and then centrifuged at 4° C. at 12000 rpmfor 5 min. The wet cells are collected (resting cells, used forhydrolysis). The wet cells were washed with physiological saline twice,mixed well, and the cell solution was analyzed with SDS-PAGEelectrophoresis. The results are shown in FIG. 2.

Example 2

Catalysis with Nitrilase Aa-Nit-Containing Resting Cells

The optimal pH, temperature, pH stability and substrate tolerance wereinvestigated.

Reaction mixture (10 mL) was composed of buffer solution (10 mL,buffer), racemic IBSN (substrate), and wet resting cells (catalyst). Thesubstrate's concentration was 0.4 mol/L. The catalyst quantity was 20 gof wet resting cells/L. The resting cells contained 70-90% of water. Thereaction was initiated in a water bath shaker at 150 rpm for 0.5 hr andterminated with 2M HCl. The conversion rate was obtained with gaschromatography to ascertain the catalytic activity of the resting cellsunder various conditions.

(1) Determination of optimal pH. With the catalysis system definedabove, the conversion rate of racemic IBSN was determined under variouspH values (pH=5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10) at 30° C.with substrate concentration at 0.4 mol/L and wet resting cells at 20g/L. The buffers for various pH were acetic acid buffer (pH=5.0-6.0),sodium phosphate buffer (pH=6.0-7.2), Tris-HCl buffer (pH=7.0-9.0), andGly-NaOH buffer (pH=9.0 -10. 0).

(2) Determination of optimal reaction temperature. With the catalysissystem defined above, the reaction was carried out at varioustemperatures (25, 30, 35, 40, and 45° C.) in Tris-HCl buffer (100 mM,pH=8.0) with the substrate concentration at 0.4 mol/L and wet restingcells of 20 g/L. The conversion rate of racemic IBSN was determined.

(3) Determination of tolerance for maximal substrate concentration. Withthe catalysis system defined above, the reaction was carried out atvarious substrate concentrations (150 mM, 300 mM, 450 mM, 600 mM, 700mM, 1 M, 1.2 M, and 1.5 M) in 10 mL of Tris-HCl buffer (100 mM, pH=8.0)with wet resting cells of 20 g/L. The conversion rate of racemic IBSNwas determined.

The results show that nitrilase Aa-Nit-containing resting cells exhibithighest catalytic activity at pH 8.0, and the optimal reactiontemperature is 30° C. The maximal substrate concentration tolerated is1.2 M.

Example 3

Application of Nitrilase Aa-Nit-Containing Resting Cells

Kinetic resolution of racemic IBSN. The reaction is shown in FIG. 1. Toa Tris-HCl buffer (10 mL, 100 mM, pH 8.0) was added racemic IBSN toreach 1.2 M, and 0.5 g of wet resting cells prepared in Example 1. Themixture was shaken at 30° C. for 15 hr in a water bath shaker. A 500 μLsample was taken every 3 hr and the reaction was quenched with 200 μL of2 M HCl. The mixture was extracted with 800 μL of ethyl acetate, shaken,and centrifuged (12000× g, 2 min). The supernatant was dried withanhydrous sodium sulfate and analyzed with gas chromatography.

(1) Determination of conversion rate and ee value with chiral gaschromatography. The amount of substrate and product present in theextract was determined with chiral gas chromatography (GC-14 C,Shimadzu, Japan). The capillary tube was BGB-174 (BGB Analytik,Switzerland). Gas chromatography conditions are below:

Sample amount 1 μL Inlet and detector temperature 220° C. Columntemperature 160° C. Carrier gas Helium Flow rate 1.6 mL/min Split ratio30:1The conversion rate and ee value were calculated according to literaturemethod reported by Rakels et al. (Enzyme Microb Technol, 1993, 15:1051).

(2) Isolation and purification of II. After the reaction, the reactionmixture is centrifuged to remove E. coli. The supernatant is evaporatedto ⅓ of the original volume.

The temperature was maintained at 80° C. for 40 min to denature theproteins before the removal of the denatured proteins throughcentrifugation. The supernatant was vacuum filtered to remove moreproteins. The filtrate was extracted with ethyl acetate (2× volume). Theaqueous layer is acidified with 2 M HCl to pH 4.0. Extraction with ethylacetate (2× volume), followed by evaporation of ethyl acetate onrotavap, affords II as an oil (ee>99.5%).

The results show that the resting cells containing nitrilase Aa-Nit cankinetically resolve IBSN at 1.2 M with the conversion rate at 42% in 15hr and ee_(p)>99%. Thus, nitrilase Aa-Nit catalysis disclosed in thecurrent invention provides a mild method to manufacture II with highconversion rate and optical purity.

It is understood that the working examples are only for illustration sothat those skilled in the art would understand the current invention andbe able to reduce the current invention to practice. These examples arein no way to limit the scope and extent of the current invention. Anyequivalent modifications or changes based on the current inventionshould be covered by the current invention.

REFERENCES CITED

-   Enzyme Microb Technol, 1993, 15: 1051-   Org. Process Res. Dev. 2008, 12: 392-398-   Angew. Chem. Int. Ed. 2008, 47: 3500-3504-   J. Mol. Catal. B: Enzym. 2006, 41: 75-80-   WO2005100580

1. An Arabis alpina nitrilase comprising the amino acid sequence shownin SEQ ID No.
 1. 2. A nitrilase encoding gene comprising the nucleosidesequence shown in SEQ ID No.
 2. 3. A recombinant vector comprising thenitrilase encoding gene of claim
 2. 4. A recombinant bacterial straincomprising the recombinant vector of claim
 3. 5. A method of producing(S)-3-cyano-5-methylhexanoic acid comprising hydrolysis of racemic2-isobutylsuccinimide by the nitrilase of claim
 1. 6. The method ofclaim 5, wherein a nitrilase gene-containing, recombinant bacterialstrain is fermented and grown to provide wet cells comprising thenitrilase
 7. The method of claim 6, where the amount of nitrilasegene-containing, recombinant bacterial strain used for hydrolysis is 50g/L based on the weight of the wet cells.
 8. The method of claim 6,wherein (a) the nitrilase gene-containing recombinant bacterial strainis inoculated into a liquid LB broth containing 50 μg/mL of kanamycinand is grown for 12 hr at 37° C.; (b) the LB broth of step (a) isinoculated into fresh liquid LB broth containing 50 μg/mL of kanamycinat 2% (v/v); (c) the nitrilase gene-containing, recombinant bacterialstrain is grown to cell concentration (OD₆₀₀) 0.4-0.8 at 37° C.; IPTG isadded to the LB broth of step (b) until the concentration reaches 0.2mM, (d) the nitrilase-gene containing, recombinant bacterial strain isinduced to grow at 28° C. for 12 hr; the LB broth of step (c) iscentrifuged at 4° C. at 12000 rpm for 5 min; and (e) the wet cells arecollected.
 9. The method of claim 6, wherein hydrolysis of racemic2-isobutylsuccinimide takes place at a pH 5.0-10.0 buffer, at 25-45° C.while stirring at 150 rpm
 10. The method of claim 6, wherein(S)-3-cyano-5-methylhexanoic acid is isolated and purified from thereaction mixture following hydrolysis.
 11. The method of claim 7,wherein the concentration of racemic 2-isobutylsuccinimide is 0.15-1.5mol/L.